But to investigate these and other mysteries about cephalopods, scientists need to be able to do genetic research on them. This requires three key elements: the complete genetic code of an organism, the ability to manipulate that code, and the ability to grow the organism in the laboratory.
For decades, this has been possible in mice and other classic model organisms, such as fruit flies and nematode worms, enabling countless advances in biology and medicine. But cephalopods – with their treasure trove of evolutionary quirks – have proven less conducive to genetic research (and not just because of the octopus’ notorious ability to escape reservoirs).
The difficulties encountered by the Rosenthal team in editing a single gene in a species of squid illustrate the challenges involved.
The first obstacle was the sequencing Doryteuthis pealeii’s genome, which the team needed to know where to cut, says Marine Biology Laboratory neurobiologist Carrie Albertin, who led the squid genome sequencing work. “Cephalopod genomes are large and complicated,” she says.
Where the human genome is made up of about 3.2 billion letters, or bases, the squid genome has about 4.5 billion letters, more than half of which are made up of repeating sequences. Linking these letters together, says Albertine, is like putting together a huge puzzle that represents an empty blue sky. “Every time you develop something new,” she says, “you have to find a way to overcome the weird challenge that biology decides to throw at you. “
After a costly effort to sequence these billions of squid DNA fragments and put them together, biology threw another curve ball at the team. Unlike other squids, Doryteuthis eggs have a thick, rubbery outer layer, or chorion, which cannot be easily punctured by the fragile needles used to inject the CRISPR-Cas9 molecular editing tool into the egg. It’s an embryonic operating game: if the needle doesn’t pierce far enough, the CRISPR-Cas9 won’t hit its target, but if the needle pierces too far, the egg won’t develop.
“I failed miserably for years,” says the embryologist and member of the St. Mary’s College Squid Editing Team. Karen crawford.
After much trial and error, made possible by the constant supply of squid eggs from Atlantic catches, Crawford found a way to use micro-scissors to make a slit in the chorion enough large so that the needle can pass through, but small enough to close behind the needle and leave the egg intact. “I got really good at pitting,” Crawford says.
For the first knockout, the team chose a gene responsible for pigmentation in squid. They selected the pigmentation gene because it would be easy to see if the modification worked. And he did. In September 2020, the group reported in the newspaper Current biology that the gene had been disrupted in 90 percent of the cells of the edited squid, which represents a key advance in making squid and other cephalopods suitable for genetic research. While the unmodified squids were speckled with colored chromatophores, the knockout squids were crystal clear.
Since then, the group has experimented with removing other genes, Rosenthal says, such as the two genes that enable RNA editing. While the function of this genetic trick is not yet clear, it appears to be essential for squid: larvae lacking RNA-editing genes die soon after hatching.
This summer, the group focused on adding, or “introducing” a gene into the squid to produce a protein that fluoresces green when it binds to calcium, which flows into the squid. axon when a nerve is triggered. Combined with the pigmentation knockout, this would allow researchers to literally observe the development of nerves and start working in transparent squid.
Despite these advances in Dorytheuthis pealeii research, and the species’ distinguished career in the service of science, the squid has a serious drawback as a genetic research organism: it cannot be easily cultivated in the laboratory. “He’s such a big adult,” Crawford says. “And he likes deep, cold sea water.”